Transient receptor potential canonical type 6 (TRPC6) O-GlcNAcylation at Threonine-221 plays potent role in channel regulation

Summary Transient receptor potential canonical type 6 (TRPC6) is a non-voltage-gated channel that principally conducts calcium. Elevated channel activation contributes to fibrosis, hypertrophy, and proteinuria, often coupled to stimulation of nuclear factor of activated T-cells (NFAT). TRPC6 is post-translationally regulated, but a role for O-linked β-N-acetyl glucosamine (O-GlcNAcylation) as elevated by diabetes, is unknown. Here we show TRPC6 is constitutively O-GlcNAcylated at Ser14, Thr70, and Thr221 in the N-terminus ankryn-4 (AR4) and linker (LH1) domains. Mutagenesis to alanine reveals T221 as a critical controller of resting TRPC6 conductance, and associated NFAT activity and pro-hypertrophic signaling. T→A mutations at sites homologous in closely related TRPC3 and TRPC7 also increases their activity. Molecular modeling predicts interactions between Thr221-O-GlcNAc and Ser199, Glu200, and Glu246, and combined alanine substitutions of the latter similarly elevates resting NFAT activity. Thus, O-GlcNAcylated T221 and interactions with coordinating residues is required for normal TRPC6 channel conductance and NFAT activation.

TRPC6 is constitutively O-GlcNAcylated at T221 to suppress basal conductance A T221A mutant TRPC6 has much greater conductance and NFATactivation Reducing but not raising O-GlcNAc alters T221dependent TRPC6 function T221 coordinates with S199, E200, and E246 to control basal channel activity INTRODUCTION Transient receptor potential canonical type-6 (TRPC6) is a non-voltage gated transmembrane cation channel conducting primarily Ca 2+ . 1 It is expressed at low basal levels in many cell types, and its upregulation and/or post-translational activation is thought to contribute to the pathophysiology of many diseases. They include glomerulosclerosis with podocyte dysfunction, 2 pathological cardiac hypertrophy, 3,4 dystrophin deficient muscular dystrophy, 5,6 wound and pathological fibrosis, 7 pulmonary vascular hypertension, 8-10 wound healing 7,11 and autoimmune disease. 12 This pleiotropic behavior has led to growing interest in TRPC6 as a pharmacological target, and selective oral inhibitors have been developed 13 and are in human trials for renal disease. TRPC6 is primarily stimulated by G-protein coupled receptor signaling via diacylglycerol and phospholipase C, 1 and by mechanical stretch. 5 Calcium conducted by TRPC6 stimulates calcium-calmodulin activated phosphatase calcineurin (CaN) to dephosphorylate nuclear factor of activated T-cells (NFAT). This results in NFAT nuclear translocation and altered gene expression. 14,15 NFAT consensus sequences within the TRPC6 promoter provide a positive feedback loop that amplifies TRPC6 associated signaling. In humans, TRPC6 gain of function (GOF) mutations result in familial segmental glomerulosclerosis. [16][17][18] GOF mutations reside in both cytoplasmic N-terminus ankyrin repeat and C-terminus domains that are physically fairly close, 19,20 and can function by disrupting inhibitory calcium binding sites. 21 TRPC6 conductance and its associated NFAT activation are normally controlled by post-translational modifications. [22][23][24][25] For example, oxidant stress coupled to NADPH oxidases NOX2 and NOX4 stimulates TRPC6 signaling. 26,27 N-glycosylation at N472 and N561 28 and phosphorylation at S14 23 are required for normal TRPC6 membrane localization and activity. Phosphorylation can both increase (at T487 by calcium-calmodulin activated kinase, CaMKII), 29 or decrease (at T70 and S262 by cGMP-activated kinase, cGK1a) 30,31 channel conductance and associated TRPC6 signaling. Hyperglycemia is also associated with TRPC6 and NFAT upregulation in kidney, [32][33][34] monocytes, 35,36 and platelets. 37 This latter observation first led us to speculate TRPC6 may also be modified by O-linked b-N-acetyl glucosamine (O-GlcNAc). [38][39][40] O-GlcNAc-ylation Here, we tested whether TRPC6 is modified by O-GlcNAcylation, and if so where this occurs and what its targeted residues impact. Proteomics identified three TRPC6 residues in the N-terminus to be constitutively O-GlcNAcylated, of which T221 conferred potent basal suppression of channel conductance and corresponding NFAT activity. Threonine 221 resides in the fourth ankyrin repeat domain, and modeling analysis found its O-GlcNAcylation enhances coordination with neighboring residues at Ser199, Glu200, and Glu246 that contribute to the constraint of basal TRPC6 conductance and NFAT signaling. The findings identify a new critical regulatory region of the protein that may help future design of pharmacological modulators.

TRPC6 is constitutively O-GlcNAcylated in the N-terminus cytoplasmic domain
To examine if TRPC6 is O-GlcNAcylated, HEK-293T cells were transfected with plasmid expressing a TRPC6-YFP fusion protein to facilitate detection of expression and provide a robust epitope for immunoprecipitation (IP). IP was performed using either YFP or O-GlcNAc as bait, and the precipitate then probed with anti-GFP (works as anti-YFP) or monoclonal anti-O-GlcNAc antibodies. The pull-down revealed that recombinant TRPC6 ($130 kD) is constitutively O-GlcNAcylated ( Figures 1A and 1B). Here the control was pcDNA. To confirm YFP itself was not the O-GlcNAc target, we repeated the experiment in cells expressing YFP alone or TRPC6-YFP. Only TRPC6-YFP was found in the oGlcNAc IP lysate ( Figure 1C).
TRPC6 is known to be constitutively N-glycosylated, and to confirm that this was not influencing the O-GlcNAc signal, we repeated the experiment using lysates pre-incubated with PNGaseF to remove this modification. The molecular weight was lower and double-band appearance was less after PNGaseF treatment as expected 47 ( Figure S1). However, the TRPC6 protein was still bound by antibodies recognizing O-GlcNAc ( Figure 1D). To test if the basal levels of TRPC6 O-GlcNAcylation could be augmented by stimulating OGT, we treated cells with TMG ( Figure 1B) but found no change in the O-GlcNAc band intensity. Additional support for the targeting of TRPC6 by O-GlcNAcylation is provided by finding co-immunoprecipitation of O-GlcNAc transferase (OGT), the enzyme that generates this modification, with YFP in cells expressing TRPC6-YFP ( Figure 1E). Lastly, we performed a Click-it TM assay in HEK-293T cells, in which O-GlcNAcylated protein residues were first selectively labeled by tetra-acetylated azide-modified N-acetylglucosamine (GlcNAz) by Y289L-b4-Gal-T1, and then co-tagged with biotin alkyne by click chemistry. The presence of biotinylation was confirmed by streptavidin-based immunoblot (upper gel), but TRPC6-YFP was only detected in cells treated with Y289L-b4-Gal-T1, further supporting TRPC6 as constitutively O-GlcNAcylated ( Figure 1F).
To directly identify O-GlcNacylation of TRPC6 and the residues involved, we performed tandem mass spectrometry. Cell lysates from HEK-293T cells over-expressing TRPC6-YFP were subjected to pull-down using anti-YFP antibody magnetic beads. The immunoprecipitant was subjected to in-gel digestion, and the digests analyzed by nanoUPLC-MS/MS analysis. Three O-GlcNAc-containing peptides in TRPC6 were identified and the modified residues mapped to Ser13/14, Thr70 and Thr221 ( Figures 1G and 1H). Of these, T221 had not been previously reported as being modified by any PTM, whereas T70 was a known phosphorylation target of cGK1a 30 and S14 by cdk5c (Liu et al., 2020). 25 T221 in particular resides in the AR4 domain of TRPC6 and is highly conserved across species ( Figure S2).

T221 O-GlcNAcylation is a primary regulator of basal TRPC6 function
To investigate the functional roles of the three O-GlcNAcylated residues in TRPC6, we performed sitedirected mutagenesis substituting an alanine at each site to prevent the PTM. Compared to wild-type (WT) TRPC6, S14A and T70A mutants modestly lowered basal NFAT activity by À19 and À36%, respectively (both p < 0.002). In contrast, the T221A mutation displayed markedly increased promoter activity (11-fold over WT, p < 10 À13 , Figure 2A). When all three mutations were introduced, NFAT activity rose further than with T221A alone ( Figure 2B). This identifies T221 as the dominant regulating site and shows influences from the other two sites depends on T221 status.

OPEN ACCESS
The prominent impact of the T221A mutation could potentially be due to intrinsic changes in the channel function from the mutation independent of its blocking O-GlcNAcylation. To address this, we transfected cells with WT or T221A TRPC6-YFP, performed IP using O-GlcNAc-Ab, and then probed the precipitate for TRPC6-YFP. As before, we observed a signal when the WT channel was expressed but little with the T221A mutant ( Figure 2C). In a second experiment, similar cells were incubated with either the OGT-inhibitor (OSMI-1) to reduce O-GlcNAcylation, or vehicle control, and NFAT promoter activity measured ( Figure 2D). Here, data are displayed normalized to control for each TRPC6 genotype to facilitate their comparison. In WT-TRPC6 expressing cells, OGT inhibition enhanced NFAT promoter activity, whereas this was blocked in cells expressing T221A-TRPC6 (p = 0.0004 for difference in OGT-inhibitor effect between genotypes by two-way ANOVA). . TRPC6 is constitutively OGlcNAcylated (A)HEK-293T expressing TRPC6-YFP or pcDNA were subjected to immunoprecipitation (IP) using O-GlcNAc antibody and then immunoblotted (IB) using GFP antibody (detects YFP-labeled TRPC6). Upper gel shows input, lower IP. (B) Same experiment but using YFP for the IP, and O-GlcNAc for IB. In this study, a group of cells were also exposed to 10 mM Thiamet G (TMG) for 24 h to assess if enhancing O-GlcNAcylation altered the signal associated with TRPC6. (C) Similar experiment using YFP as the control. The input shows either YFP or TRPC6-YFP expressed. After IP for OGlcNAc, IB for YFP shows only in the TRPC6-YFP band region. We next tested whether the T221A mutation disrupted TRPC6 membrane expression or its external structure, using a potent selective TRPC6 antagonist (BI 749327) 13 and found this inhibitor significantly reduced NFAT promoter activity in both WT TRPC6 and T221A mutant ( Figure 2E). This indicates TRPC6-T221A  iScience Article membrane localization and external structure were likely intact. By contrast, N-glycosylation is required for normal membrane localization. 28 To determine the functional impact of T221A mutagenesis, TRPC6 calcium conductance was measured by patch-clamp assay in HEK-293T expressing WT or T221A TRPC6. Figure 2F shows typical raw currentvoltage-time tracings, and Figure 2G the summary data. Bidirectional voltage-dependent current increased by 75-80% in cells expressing T221A-TRPC6 versus WT. This is similar to changes in current induced by angiotensin stimulation of TRPC6 conductance. 30 Here we also compared the changes in current density-voltage dependence with T221A to a well described GOF mutation P112Q associated with aggressive renal disease. 16 At positive voltages (outward current), the P122Q mutation fell intermediate between WT and T221A, whereas at negative voltages (inward current), P122Q had no impact, whereas this was still markedly increased by T221A ( Figure 2G). These data identify T221 as a bidirectional regulator of basal TRPC6 Ca 2+ current.
Lastly, we examined the impact of T221 on resting and agonist stimulated NFAT signaling in cardiomyocytes that normally express low levels of TRPC6 but on its activation display pathological hypertrophic signaling. 30 Myocytes were transfected with either WT or T221A TRPC6 achieving a high relative level of Trpc6 expression mostly reflecting the very low baseline. We observed increases in two prominent biomarkers of hypertrophic signaling, Nppa (A-type natriuretic peptide) and Rcan1 (regulator of calcineurin-1) in cells expressing the T221A mutant, whereas Nppb (B-type natriuretic peptide) rose similarly with both ( Figure 2H). We also tested if expression of T221A in myocytes limits or amplifies enhancement of NFAT signaling induced by angiotensin II stimulation. As before, myocytes expressing T221A TRPC6 exhibited increased expression of both Nppa and Rcan1, but AII stimulation still augmented this further and similarly as in cells with WT TRPC6 ( Figure 2I). There was no no significant interaction (2W-ANOVA) between AII or genotype effects. Thus, mutating T221 does not modify channel activation by canonical Gq-coupled DAG-activation.

Homologous threonines to T221 in related TRPC3 and TRPC7 confers similar activity control
Among the seven-member TRPC channel family, TRPC3, TRPC6, and TRPC7 form a closely related subgroup, with TRPC3 and TRPC6 being most homologous. 20 We therefore tested if the functional role of T221 in TRPC6 was shared by homologous threonines in the other channels. Sequence analysis identified T150 in TRPC3 and T166 in TRPC7 as homologous to T221 in TRPC6 ( Figure S3). Each of these were then mutated to alanines, expressed in HEK cells, and the impact on NFAT promoter activation determined (Figure 3A). Each T/A mutation was associated with a near 10-fold rise in NFAT promoter activity over the respective WT channel. These data support conservation of this regulatory region in the structure of all three members of this TRPC subfamily.

Independence of TRPC6-T221A NFAT activation on GOF mutations or hyperglycemia
Based on our preceding findings, we speculated that gain-of-function TRPC6 mutations that elevate basal NFAT activity may work in part by interfering with T221 O-GlcNAcylation. To test this, vectors expressing several known potent GOF mutations (P112Q, M132T, and R175Q) that amplify resting NFAT promoter activity were studied ( Figure 3B). None of these augmented NFAT promoter activity as much as T221A (e.g., 1-2x versus 11x). If the mutations partially interfered with T221 O-GlcNAcylation, we anticipated by mass action that enhancing it with TMG might lower NFAT activity due to the mutation. However, this was not observed with any of the mutations ( Figure 3B). Lack of interaction was further tested by expressing T221A, P112Q, and their combination. All three increased NFAT activity, but T221A alone or combined with P112Q achieved the same level ( Figure 3C). This supports the conclusion that such GOF mutations unlikely interfere with T221 O-GlcNAcylation as a mechanism for their NFAT activation.
Our initial impetus for exploring O-GlcNAcylation of TRPC6 was triggered a hypothesis that this PTM might be linked to NFAT activation by hyperglycemia (HG). As displayed in Figure 3D, HG stimulates NFAT promotor activation particularly in HEK293 cells expressing TRPC6 (mannitol is used as a negative control). Our results had shown that O-GlcNAcylation reduced NFAT activation, so the notion this was a mechanism of diabetic TRPC6 signaling was refuted. Here we further tested this independence by increasing total protein O-GlcNAcylation by 200-300% with either HG, TMG, or glucosamine, and comparing their impact on NFAT activation. (Figures 3E and 3F). The latter was ony significantly increased by HG despite near identical levels of augmented O-GlcNAcylation by all three interventions ( Figure 3G).  iScience Article and hydrogen bond formation between the side chain of glutamine (Q198, pink) and the backbone amino group of aspartate (D205, light blue). We posited that these interactions hold a portion of the 193-203 loop in place to enhance S199 and E200 for interaction with O-GlcNAc at T221. The side chains of S199 (yellow), E200 (blue), and E246 are predicted to form hydrogen bonds with the hydroxyl of O-GlcNAc.
To test this, we selectively mutated each residue to an alanine, expressed the recombinant forms of TRPC6 in HEK-293T cells, and assessed NFAT activity. Figure 4B shows results for NFAT activity. E246A and S199A mutations similarly increased NFAT activity over WT, but both less than with the T221A mutation. Neither Q198A nor E200A alone augmented activity over WT, however when both S199A and E200A were combined, the result was comparable with T221A. Adding E246A to form a triple mutant did not further increase NFAT activity. Figure S4 shows an example immunoblot for protein levels obtained with each mutant transfection, with slight changes in some but far less than their impact on corresponding NFAT activity. Thus, the coordinating hydrogen bonds between S199, E200, and O-GlcNAcylated T221 form critical residue interactions regulating basal TRPC6 conductance and NFAT-activation, with E246 itself also impacting this behavior.

TRPC6 T221 O-GlcNAcylation enhances protein longevity
Beyond stimulating TRPC6 expression, channel conductance and NFAT signaling, we tested if T221 O-GlcNAcylation stabilizes the protein to protect it from proteasomal degradation. T221A or WT TRPC6 were expressed in HEK-293T cells exposed to cycloheximide to block de novo protein synthesis +/À the proteasome inhibitor MG132. The rate of protein decline was indexed by GFP fluorescence and was significantly iScience Article faster with the T221A mutant ( Figure 4C). This primarily reflected increased proteasome degradation as MG132 restored levels to baseline similarly with the mutant and WT form ( Figures 4D and 4E). Thus, O-GlcNAcylation of TRPC6 reduces its proteasomal degradation to improve post-translational longevity.

DISCUSSION
In this study, we have identified three amino acids in the AR4-LH1 region of the N-terminus of TRPC6 that are constitutively modified by O-GlcNAc, and find that among these, T221 exerts the dominant control over basal channel current and associated NFAT activation. Preventing O-GlcNAcylation at this residue using a T221A mutation results to our knowledge in the highest basal channel conductance and associated NFAT activity yet reported, including from human GOF mutations that cause renal disease or by other activating PTMs. Consistent with its augmentation of NFAT promotor activity, expression of mutant T221A-TRPC6 amplifies cardiomyocyte pathological hypertrophic gene programs while leaving further receptor-operated (Gq-GPCR) stimulation intact. Although the molecular mechanism for T221A GOF remains to be determined, it appears independent of that associated with GOF mutations. We also find O-GlcNAcylation at T221 coordinates with neighboring amino acids required to constrain channel conductance at normal levels, revealing this region as a key regulatory nexus for the channel. Although we had first hypothesized that TRPC6 O-GlcNAcylation might be a mechanism for HG-stimulated TRPC6-dependent NFAT activation, our data refute this by showing that this modification is already present and that reducing not increasing it results in NFAT activation. These results identify a novel regulatory region of the protein that might be leveraged for therapeutics to constrain hyperactive TRPC6 and associated disease.
It is useful to frame our findings in the context of recent cryo-EM structural data regarding TRPC6. 19,20,49 Such studies reveal TRPC6 tetramers in a two-layered architecture assembled into an inverted bell-shaped intracellular cytosolic domain (ICD) that caps below the transmembrane domain (TMD). The ICD is assembled through interactions between the four ankyrin repeat domain (residues: 96-243) in the N-terminus, linker helices (residues: 256-393) and a coiled-coil domain in the C-terminus. ARs and LHs are key to inter-subunit interactions and TRPC6 tetramer assembly. Specifically, amino acids of the N-terminal loop (85-94) interact with LHs of the neighboring subunit and the last three LHs pack against the TRP helix providing the major contact site between the ICD and TMD. The ARs are highly conserved helix-turn-helix structural motifs 50-52 involved in the assembly and stability of multiprotein complexes by forming both intra-and inter-repeat hydrophobic and hydrogen bond interactions. 53,54 We find O-GlcNAcylated T221 forms stabilizing electrostatic contacts between AR4, the 193-203 loop near AR4, and loop connecting AR4 to LH1. We believe this helps hold distant regions together and is critical to stabilize the closed state of the channel pore. Many GOF mutations causing renal disease reside in ARs: G109S (AR1), P112Q (AR1), N125S (AR1), M132T (AR2), N143S (AR2), R175Q(AR3), whereas others are found in the C-terminus (e.g. Q899K, R895C, E897K) that is structurally proximate. 20, 48 Structural studies of these mutants similarly support destabilization of electrostatic interactions at the interface of AR domains and the linker helix that in turn associates with greater current. 49 A unifying impact of these mutations appears to be suppression of allosteric inhibition by intracellular calcium. 21 Although our data indicate some of these mutations do not prevent T221 O-GlcNAcylation, it remains possible others may, though this seems unlikely or their impact would be so large as to be embryonically lethal. Indeed, TRPC6 T221 mutations have not been reported in humans. Regardless, identification of the residue cluster T221-O-GlcNAc, S199, E200, and E246 and Q198 supports it being a key conserved region required for channel function, linked to constitutive T221 O-GlcNAcylation.
Although attention to O-GlcNAc modifications has often been on its role in disease, it is also known to play constitutive roles. 41,55,56 It is among the most abundant forms of protein glycosylation, 57 with OGT found in all metazoans and expressed in all mammalian tissues. 58,59 OGT also has non-catalytic functionality, but only the O-GlcNAcylation function of OGT appears required for cell survival. 60 OGT strongly associates with the ribosome and nearly half of ribosomal proteins are O-GlcNAcylated. 61 For example, O-GlcNAcylation of Sp1 and Nup62 occurs co-translationally and is key to their stability, 62 whereas that of nuclear pore proteins is required for their functionality. 63 O-GlcNAcylation is also used to modulate function, as in the case of phospholamban at Ser16 to inhibit myocyte Ca 2+ uptake by the sarcoplasmic reticulum, 64 STIM1 where it impedes store-operated Ca 2+ entry that could influence mechanical and receptor-coupled signaling, 65 and calcium-calmodulin stimulated kinase II at Ser279 that activates the enzyme. 66 Although we did not find evidence that further enhancement of constitutive TRPC6 O-GlcNAcylation has biological effects, its reduction caused channel activation. Of the reported post-translational modifiers of TRPC6, only N-glycosylation and from the current results O-GlcNAcylation appear required for normal channel localization and conductance. iScience Article Although the current results remove TRPC6 O-GlcNAcylation as a mechanism for its increased activity in hyperglycemic conditions, they do provide a strong example of where O-GlcNAc modifications are required for normal protein function. The data also specifically reveals a previously unknown yet highly conserved regulatory region, sharing homology with closely related channels TRPC3 and TRPC7, and this new insight may help provide new pathways for therapeutics to modify channel function.

Limitations of the study
Although site mutagenesis is commonly used to study the functional impact of targeted post-translational protein modifications, and alanine substitutions will prevent O-GlcNacylation at a given site, it has limitations. Specifically, one may observe changes that are because of substituting the native amino acid to alter protein structure/function itself. Although we cannot fully rule out this possibility we believe the data support a primary effect related to prevention of O-GlcNAcylation. First, we find reducing O-GlcNAcylation with OGT inhibition also increases O-GlcNAcylation of TRPC6 and NFAT activation, yet has significantly less impact on either behavior when the T221A mutation is expressed. Second, the modeling analysis predicts local residue interactions based on T221A being O-GlcNAcylated and not just mutated to alanine, and these predictions are experimentally confirmed. The molecular weights for TRPC6-YFP in our IP gel studies are close to predicted, but did differ somewhat from gel to gel. In particular, we found the weights of the immunoblot from the IP lysates a bit lower than in the input material. This may reflect loss of some of the PTMs on the protein such as N-glycosylation. Still, finding TRPC6 O-GlcNAcylation by IP assay, mass spectrometry, and click-chemistry assay, and reduced changes in the T221A mutation supports this as directly modifying TRPC6. Lastly, the mechanism by which T221 OGlcNAcylation and its interactants control channel conductance and associated NFAT activity remains to be determined, but maybe identified by a future cryo-electron microscopy analysis.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

Electrophysiology studies -Patch clamp
Patch clamp studies were done as previously described protocol (Galvis-Pareja et al., 2014 71 ). Briefly, HEK-293T cells were plated in glass coverslips in a 24 well plate and transfected with 1 mg of TRPC6 channel plasmids (WT, T221A and P112Q) using Xfect transfection reagent according to manufacturer's protocol (Takara Bio 631318). Cells expressing wildtype and mutant channels were identified by YFP fluorescence. The bath solution was 140mM NaCl, 5mM CsCl2, 1 mM MgCl2, 10 mM HEPES, and 10 mM glucose with pH of 7.4. Borosilicate glass capillary pipettes (World Precision Instr.) were used with $3MU resistance when filled with solution containing 5mM NaCl, 40 mM CsCl2, 80mM Cs-glutamate, 5mM Mg-ATP, 5 mM EGTA, 1.5 CaCl2 (free calcium concentration was 100 nM). Currents were obtained using a voltage step-pulse protocol or ramp protocol from -100 mV to +100 mV applied every 2s for 500 ms from holding potential of -60 mV. Current recordings were in a whole-cell configuration using Axopatch 200A amplifier (Axon Instruments, Molecular Devices).

Neonatal Rat ventricular myocyte isolation and adenoviral transfection
Neonatal Rat cardiomyocytes (NRVMs) were freshly isolated as previously described (Mishra et al., 2021 72 ) and cultured at 1 million cells per well in six-well plates for 24 h in DMEM with 10% FBS and 1% penicillin/ streptomycin. Adenoviruses were developed expressing the GFP-tagged wild-type sequence of human TRPC6 or TRPC6 T221A. NRVMs were infected with an MOI of 10 with the respective viruses for 48 h before performing the downstream assays. iScience Article intensities of the wells were measured at the indicated time points. T221A-GFP decline was measured in cycloheximide treated cells in the presence or absence of proteasome inhibitor MG132 (10 mM). For western blot experiments, cells were plated in 6 well dishes and transfected with 5mg plasmids per well. 24 h after transfection, HEK cells were incubated with 100 mg/mL of cycloheximide. Cells were harvested using100 mL RIPA cell lysis buffer at the indicated time points as shown in Figure 4D, followed by SDS-PAGE and western blotting to visualize TRPC6 T221A protein levels.

Statistical analysis
Statistical analysis was performed using Prism Ver 9.3.1. All of the individual tests used for each datafigure in the study are provided in their respective figure legend along with sample size per group. For multiple groups, 1-way ANOVA, a Welch ANOVA (if test for variance difference between groups was positive), or non-parametric Kruskal Wallis (if non-normally distributed) was used. A 2-Way ANOVA was also used in some testing as indicated. Two-group comparisons used non-parametric tests (Mann Whitney U test). All precise p values are provided for statistical testing in the figures and/or legends.